【作者】 高明辉；

【导师】 马立保；

【作者基本信息】 华中农业大学 ， 动物营养与饲料科学， 2008， 硕士

【摘要】 为了研究亚硝酸盐胁迫对异育银鲫的毒性效应以及在日粮中添加高水平的维生素C(VC)、维生素E(VE)是否能够提高异育银鲫抵抗亚硝酸盐的胁迫能力,本文通过两个试验对此进行了研究。1.亚硝酸盐对异育银鲫血液指标的影响试验一选取平均体重为37.59±3.29g的健康异育银鲫150尾,亚硝酸盐浓度设3个浓度梯度组(0.05mg/l、0.32mg/l、1.28mg/l),每个处理组5个重复,每个重复10尾,暴露时间为48h和14d,通过对异育银鲫血液参数的检测来研究亚硝酸盐对异育银鲫的毒性作用。试验一结果显示:亚硝酸盐浓度和暴露时间对血清LDH活性具有显著地影响,且两者具有明显地交互作用(P＜0.01)。在暴露48h和14d时,1.28mg/l的亚硝酸盐组血清谷丙转氨酶(GPT)、谷草转氨酶(GOT)、乳酸脱氢酶(LDH)活性均显著地高于0.05mg/l组(P＜0.05),并高于0.32mg/l的亚硝酸盐暴露组。暴露浓度和时间对血清尿素水平具有显著地交互作用(P＜0.01)。在暴露48h时,随着亚硝酸盐浓度的增加,异育银鲫血清尿素含量显著地上升(P＜0.05),而在暴露14d时,1.28mg/l组血清尿素水平与0.05mg/l组相比显著地降低(P＜0.05)。亚硝酸盐浓度和暴露时间对血浆K~+的水平具有明显的交互作用(P＜0.05),且亚硝酸盐浓度对血浆K~+水平影响较大。0.32mg/l和1.28mg/l的亚硝酸盐暴露48h可以显著地降低血浆Cl~-水平,而随着暴露时间的延长,血浆Cl~-水平几乎不受影响,且亚硝酸盐浓度和暴露时间对血浆Cl~-、Na~+水平均无交互作用。试验一结果表明,亚硝酸盐暴露使异育银鲫部分血液生化指标发生显著地变化,通过对血液生化指标的检测可以反映出亚硝酸盐对鱼体组织,特别是肝胰脏、鳃等组织的毒性作用以及水域的污染情况;亚硝酸盐暴露能够引起异育银鲫肝细胞的损伤和组织缺氧,并可能导致异育银鲫代谢方式的转变以适应厌氧生活;亚硝酸盐暴露可能影响氮的代谢,使尿素的生成增加;亚硝酸盐暴露似乎不能引起异育银鲫血浆Na~+的不平衡。2.VC、VE对亚硝酸盐胁迫下异育银鲫血液指标、抗氧化能力及组织HSP70表达的影响试验二选择平均体重为251.04±45.02g的异育银鲫168尾,随机分为4个组(C组,N组,N+HE组,N+HC组),每组7个重复,每个重复6尾,分别饲喂3种日粮(Ⅰ,Ⅰ,Ⅱ,Ⅲ型),试验期为21d。通过对异育银鲫血液生理生化指标、抗氧化指标的测定及肝胰脏和肾脏中热休克蛋白70家族(HSP70)的表达来反映亚硝酸盐胁迫对异育银鲫的毒害作用及高水平VC、VE的抗胁迫效应。试验得出如下结论:0.85mg/l的亚硝酸盐胁迫21d使异育银鲫红细胞(RBC)数量和红细胞压积(HCT)显著地上升(P＜0.05),使血红蛋白(Hb)含量有所下降;日粮中添加高水平的VC(500mg/kg)、VE(350mg/kg)对亚硝酸盐胁迫下异育银鲫RBC数量、HCT均没有影响,而添加VC、VE可以使该条件下异育银鲫Hb含量恢复到接近正常水平。亚硝酸盐胁迫使血浆K~+、Cl~-的含量显著地降低(P＜0.05),而对Na~+含量没有影响;日粮中添加高水平的VE对亚硝酸盐胁迫下血浆三种离子的含量均无显著影响,而日粮中添加高水平的VC可以使亚硝酸盐胁迫下异育银鲫血浆三种离子含量恢复到接近正常水平。亚硝酸盐胁迫可以显著地提高异育银鲫血清LDH活性(P＜0.05),同时使血清GPT、GOT活性增加;日粮中添加高水平的VC、VE使暴露鱼血清LDH活性下降到接近正常水平。亚硝酸盐暴露21d可以显著地降低异育银鲫的血糖(GLU)水平(P＜0.05);而日粮中添加高水平的VC可以显著地提高暴露鱼的血GLU水平(P＜0.05),而添加高水平的VE对血GLU水平没有显著地影响。亚硝酸盐胁迫使异育银鲫血清和肝胰脏中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)活性及总抗氧化能力(T-AOC)下降,丙二醛(MDA)含量显著增加(P＜0.05);日粮中添加高水平的VC、VE可以使胁迫条件下异育银鲫血清和肝胰脏中SOD、GSH-PX活性上升及T-AOC增强,MDA含量显著降低(P＜0.05),从而提高机体的抗氧化能力。亚硝酸盐胁迫可以显著地提高异育银鲫肝脏及肾脏中HSP70的表达(P＜0.05),从而保护细胞或修复损伤细胞。日粮中添加高水平的VC、VE可以显著地降低亚硝酸盐胁迫下异育银鲫肝脏和肾脏中HSP70的表达(P＜0.05)。试验二结果表明,亚硝酸盐胁迫可以导致组织器官的损伤,使组织中特异性酶GPT、GOT、LDH释放进入血液,引起酶活性的升高;亚硝酸盐胁迫可以影响异育银鲫血浆离子含量、血糖及HSP70的表达;促氧化作用和抗氧化能力的不平衡,是亚硝酸盐胁迫对异育银鲫的毒性机制之一;日粮中添加高水平的VC、VE可以在一定程度上缓解亚硝酸盐胁迫的负面影响,从而提高机体的抗胁迫能力。更多还原

【Abstract】 Two experiments were conducted in this study to investigate the toxic effect of nitrite on Carassius auratus gibelio and whether high level vitamin C(VC),vitamin E(VE) can improve the anti-stress ability of Carassius auratus gibelio under nitrite stress.1.Effects of nitrite exposure on blood index of Carassius auratus gibelio150 healthy Carassius auratus gibelio,with the average weight of 37.59±3.29g were randomly divided into 3 groups(group 1,group 2,group 3,for 0.05,0.32,1.28mg/l nitrite, respectively) of 5 replicates of 10 caudaes each,respectively.After under the 48h and 14d’s to nitrite sodium,blood parameter were sampled and analyzed to study the toxic effect of nitrite on biochemical parameter of blood of Carassius auratus gibelio.The results shows that:The concentration of nitrite and exposure time significantly affected the serum LDH, and the significant interaction effect of exposure time and concentration was observed (p＜0.01).The activity of serum glutamate pyruvate transaminase(GPT), glutamic-oxaloacetic transaminase(GOT),lactate dehydrogenase(LDH) in group 3 were significantly higher in 48h and 14d’s exposure to nitrite,compared to control group(0.05mg/l of nitrite)(p＜0.05),and the activity of three enzymes in group 3 was higher than the group 2.Under 48h’s exposure to nitrite,serum urea significantly increased with the increasing of nitrite concentration(p＜0.05),whereas serum urea in 1.28mg/l dose group significantly decreased after the exposure of 14d,compared to control group(0.05mg/l of nitrite)(p＜0.05).The significant interaction effect between exposure time and concentration was observed(p＜0.01).The concentration of nitrite and exposure time also interacted remarkably on plasma K~+(p＜0.05),and exposure time did not significantly affected plasma K~+.Plasma Cl~-was significantly decreased after the exposure of 48h in 0.32mg/l and 1.28mg/l dose group, but plasma Cl~-was not affected by the increasing of exposure time,the concentration of nitrite and exposure time had no significant interaction on plasma Na~+,Cl~-.The result of experiment one demonstrated that some biochemical indexes in blood of Carassius auratus gibelio were significantly affected by the exposure of nitrite.Blood biochemical parameter could reflect the toxic effect of nitrite on tissue,especially the hepatopancreas,gill and water pollution;nitrite exposure could cause damage to hepatocyte and hypoxia of tissue,the way of metabolism may change to adapt the anaerobic life;Nitrite exposure may affect nitrogen metabolism,serum urea production increased;exposure to nitrite seemed not to induce plasma Na~+unbalance.2.Effects of VC、VE on blood indices,antioxidative ability and HSP70 expression in tissue of Carassius auratus gibelio under nitrite stress168 healthy Carassius auratus gibelio with the average weight of 251.04±45.02g were selected in the experiment two.The fishes were randomly divided into 4 groups (C group,N group,N+HE group,N+HC group) of 7 replicates of 6 caudaes each, respectively.The fishes of 4 groups fed 3 species of diet(Ⅰ,Ⅰ,Ⅱ,Ⅲ) respectively for 21d.Physiological and biochemical parameter in blood,anti-oxidative indexes in serum and hepatopancreas,heat shock protein 70(HSP70) expression in hepatopancreas and kidneys were determined to reflect toxic effect of nitrite on Carassius auratus gibelio and the anti-stress effect of high dose of VC,VE.The results showed that:After exposure to 0.85mg/l nitrite for 21d,the number of red blood cell(RBC) and hematocrit(HCT) significantly increased(p＜0.05),haemoglobin(Hb) decreased;During nitrite exposure,the group fed 500mg/kg VC,350mg/kg VE did not affect the number of RBC,HCT,whereas the level of Hb in the group which fed 500mg/kg VC,350mg/kg VE could recover to the normal level.K~+,Cl~-level in plasma significantly decreased(p＜0.05), plasma Na~+concentration was did not show changes.High levels of VE did not affect the plasma ion(K~+,Cl~-,Na~+) levels,whereas plasma ion levels in the group with high levels of VC could return to the normal levels.During nitrite exposure,serum LDH activity significantly increased(p＜0.05), meanwhile the activity of serum GPT,GOT increased;Serum LDH activity in both groups with high level of VC,VE almost decreased to the normal level.Serum glucose(GLU) level significantly decreased after 21d’s exposure to nitrite(p＜0.05);After exposure to nitrite,serum GLU level in the group with high levels of VC significantly increased(p＜0.05),but the level of VE did not affect GLU level.After 21d’s exposure to nitrite,the activity of superoxide dismutase(SOD) and glutathione peroxidase(GSH-PX),total antioxidative capacity(T-AOC) in serum and hepatopancreas decreased,whereas maleic dialdehyde(MDA) concentration significantly increased(p＜0.05);in the groups with high levels of VC,VE,the activity of SOD and GSH-PX,T-AOC in serum and hepatopancreas increased,whereas MDA content significantly decreased(p＜0.05),high level of VC or VE could strengthened antioxidant ability of Carassius auratus gibelio.After exposure to nitrite,HSP70 expression in hepatopancreas and kidneys significantly increased(p＜0.05),suggesting protective function on cells or repairing the damaged cells;Under the same exposure to nitrite, HSP70 expression in hepatopancreas and kidneys with high levels of VC,VE significantly decreased(p＜0.05).The result of experiment two indicated that nitrite stress caused damage to tissue, cell injury of organs,which led to release of tissue-specific enzymes into blood,causing activity of enzymes increased;Nitrite stress could affect plasma ion level,GLU,HSP70 expression;An imbalance between prooxidant forces and antioxidant defenses is one of the toxicity mechanisms of nitrite on Carassius auratus gibelio.High level of VC or VE in diet could reduce the passive influences induced by nitrite stress at some degree, increasing stress resistance of Carassius auratus gibelio.更多还原